Aldo-Keto Reductases (AKRs) of the 1A, 1B, 1C and 1D sub-families are soluble monomeric reductases that may regulate lipophilic ligand access to nuclear receptors (NR): steroid and orphan. For example, human recombinant AKR1C3 acts as a 3-, 17- and 20-ketosteroid reductase and may regulate ligand occupancy and frans-activation of the androgen, estrogen and progesterone receptor (AR, ER and PR). AKR1C3 (also known as prostaglandin F synthase) will reduce PGD2 to 11(3-PGF2 (proliferative signal) and may deprive peroxisome-proliferator activated receptor y (PPARy) of its PGJ2 series ligands (a differentiation/apoptotic signal). This pre-receptor regulation of hormone action may have profound consequences on the proliferation of prostate and breast where AKR1C3 is highly expressed. AKR1C3 inhibitors would deprive steroid receptors of their ligands and could be used to treat hormone dependent malignancies. AKR1C3 inhibitors may also lead to the activation of PPARy and could be used to treat malignancies refractory to hormone ablation. Nonsteroidal anti-inflammatory drugs (NSAIDs) are potent inhibitors of AKR1C3 suggesting it may be a non-COX target for their chemopreventive actions. We have developed NSAID analogs that do not inhibit COX-1/2 but retain inhibitory potency against AKR1C isoforms. This proposal will elaborate the role of AKR1C3 in NR signaling in hormone responsive and refractory prostate and breast cancer cells and advance AKR1C3 inhibitor development. Aim 1, will determine whether AKR1C3 metabolizes lipophilic ligands (steroids and prostaglandins) in transiently transfected null-cells and in prostate and breast cancer cells that ectopically express AKR1C3. Aim 2, will determine whether AKR1C3 changes the concentration of lipophilic ligands to alter frans-activation of the AR, ER, PR and PPARyt endogenous gene expression, and proliferative properties of hormone responsive and refractory prostate and breast cancer cell lines. Aim 3, will use immunohistochemistry and laser capture microdissection coupled with real-time PCR to measure co-localization of AKR1C3 with NR in normal and diseased prostate and breast. Aim 4, will use crystal structures of AKR1C3"NADP(H)"NSAID complexes to develop AKR1C3 isoform specific inhibitors. These studies will establish whether AKR1C3 is a non-Cox target for the cancer chemopreventive roles of NSAIDs.